A variety of molecules having affinity for particular binding sites in cells are known. For example, antibodies and cell-surface receptors can bind to specific targets. However, the strength of the interaction between a given molecule and its target will in some instances be low.
Efforts to increase the affinity of antibody fragments to their target have resulted in the production of dimerized antibody fragments, wherein the dimer has two binding sites, each of which is specific for the antibody target. Dimerization has been conducted by a variety of means, generally involving modification of a “tail” region attached to the antibody fragment. Thus, self-associating secondary structures such as helix bundles have been employed in an effort to produce dimerizable units which retain their specificity and ability to bind to a target of interest. However, the use of known self-associating domains can present several problems, including unwanted and non-functional aggregation of the molecules, as well as difficulties in obtaining optimal spacing between molecules, resulting from limits on control over the geometry of the resultant structure. It is often desirable that the binding region of each molecule in a dimer is located on the same face of the dimer, with sufficient spacing between the molecules to allow engagement of their target molecules.
Successful efforts at forming oligomers of more than two self-associating subunits in association with specific-binding regions have been limited. In one instance, a tetramer of subunits comprising a modified helix of the transcription factor GCN4 together with a “miniantibody” was produced. Similarly, the coiled-coil assembly domain of the cartilage oligomeric matrix protein fused to a small peptide has been used to form pentamers. However, the structure of the cartilage oligomeric assembly is believed to be thin and rod-shaped which may render it unsuitable for use with larger peptides or proteins for which greater inter-unit spacing may be needed.
A discussion of efforts to produce self-assembling molecules can be found in Pluckthun et al., 1997, ref. 1, as well as Terskikh et al., 1997 ref. 2.